A total of 1 × 107/ml B cell-depleted splenocytes or purified B cells resuspended in PBS were labeled with 2 μM CFSE (BioLegend) for 15 min at 37 °C and then washed twice with complete RPMI-1640 medium for culture. To monitor the polarization of B cells, murine splenic B cells were isolated and labeled with CFSE and then cultured with CD40 mAb, LPS, or CpG-ODN 1826 in the presence or absence of 0.5 mM NaBu for 48–96 h plus L + PIM for the last 5 h. To monitor T cell proliferation, splenocytes labeled with CFSE were seeded in plates precoated with αCD3 (5 μg/ml; BioLegend) and αCD28 (1 μg/ml; BioLegend) and cocultured for 72 h with B cells (splenocytes: B cells = 1:1 or 1:2), which were washed twice with PBS after induced by LPS with or without NaBu for 48 h. Cell proliferation was detected by dilution of CFSE with flow cytometry.
Carboxyfluorescein succinimidyl ester (cfse)
Manufactured by BioLegend
Sourced in United States, Germany
CFSE (Carboxyfluorescein Succinimidyl Ester) is a fluorescent dye used for cell labeling and tracking. It binds to cellular proteins, allowing the visualization and quantification of cell division and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (29 mentions)
Celltrace violet, by Thermo Fisher Scientific (18 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BD (11 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (6 mentions)
Lipopolysaccharides (lps), by Merck Group (6 mentions)
Anti cd3, by BioLegend (6 mentions)
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Facscalibur, by BD (5 mentions)
7 aad, by BioLegend (5 mentions)
Anti cd28, by BioLegend (5 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (4 mentions)
Facscanto 2, by BD (4 mentions)
Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (4 mentions)
Lsrfortessa, by BD (4 mentions)
Rhil 2, by Thermo Fisher Scientific (4 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Anti cd3 cd28 beads, by Thermo Fisher Scientific (4 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by STEMCELL (3 mentions)
Cd8a t cell isolation kit, by Miltenyi Biotec (3 mentions)
256 protocols using carboxyfluorescein succinimidyl ester (cfse)
1
CFSE-Based B and T Cell Proliferation Assay
2
T-Cell Proliferation Assay with Cisplatin-Treated A549 Cells
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Whole blood was collected from healthy individuals at the Suzhou Blood Center
(Suzhou, China) and subjected to density gradient separation on Ficoll-Paque Plus (GE
Healthcare, USA). After centrifugation, the peripheral blood mononuclear cell (PBMC)
layer was collected, seeded onto a tissue culture plate, and incubated at 37°C in a
5%-CO2 incubator. After 2-h incubation, cells in suspension were
collected following gentle pipetting the medium, and these were predominantly T
cells. The isolated T cells were labeled with carboxyfluorescein succinimidyl ester
(CFSE; Biolegend) as previously described (14 (link)). Meanwhile, A549 cells were treated with cisplatin (25 mg/mL; Biolegend)
for 3 h. The CFSE-labeled T cells were then seeded into 96-well plates
(2×105 cells/well) that had been pre-coated overnight with anti-CD3 (5
µg/mL, Biolegend) and anti-CD28 (2.5 µg/mL, Biolegend) at 4°C. The cisplatin-treated
A549 cells with or without B7-H1 blocking antibody (50 µg/mL, Biolegend) were then
added to CFSE-labeled T cells at a T:A549 ratio of 1:2, 1:4, or 1:8. Each condition
was tested in triplicate. After 72 h, all cells were collected and T-cell
proliferation was examined by flow cytometry.
(Suzhou, China) and subjected to density gradient separation on Ficoll-Paque Plus (GE
Healthcare, USA). After centrifugation, the peripheral blood mononuclear cell (PBMC)
layer was collected, seeded onto a tissue culture plate, and incubated at 37°C in a
5%-CO2 incubator. After 2-h incubation, cells in suspension were
collected following gentle pipetting the medium, and these were predominantly T
cells. The isolated T cells were labeled with carboxyfluorescein succinimidyl ester
(CFSE; Biolegend) as previously described (14 (link)). Meanwhile, A549 cells were treated with cisplatin (25 mg/mL; Biolegend)
for 3 h. The CFSE-labeled T cells were then seeded into 96-well plates
(2×105 cells/well) that had been pre-coated overnight with anti-CD3 (5
µg/mL, Biolegend) and anti-CD28 (2.5 µg/mL, Biolegend) at 4°C. The cisplatin-treated
A549 cells with or without B7-H1 blocking antibody (50 µg/mL, Biolegend) were then
added to CFSE-labeled T cells at a T:A549 ratio of 1:2, 1:4, or 1:8. Each condition
was tested in triplicate. After 72 h, all cells were collected and T-cell
proliferation was examined by flow cytometry.
3
PDAC Organoids Potentiate CD8+ T Cell Killing
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Mouse PDAC cells stably expressing OVA and carrying doxycycline (Dox)-inducible mTurquoise2-Atg4BC74A were grown as organoids, treated with or without Dox (1 μg/mL) for 96 hrs. Organoids were dissociated into single cells, which were then incubated with either anti-H-2Kb-SIINFEKL antibody (clone 25-D1.16, BioXCell, BE0207) or isotype control (clone MOPC-21, BioXCell, BE0083) at 100 μg/mL for 30 min at 4 °C. Total splenocytes were harvested from OT-I mice and CD8+ T cells were enriched using Dynabeads® Untouched™ Mouse CD8 Cells (Invitrogen, 11417D) following manufacturer’s instructions. Isolated CD8+ T cells were labelled with 10 μM CFSE (BioLegend) for 10 min at RT in the dark, washed 3x with RPMI-1640 supplemented with 10% FBS. Ten thousand PDAC cells and forty thousand CD8+ cells were seeded in 96-well plates and cultured in 100 μL 50% DMEM and 50% RPMI-1640 supplemented with 10% FBS, 10 ng/mL recombinant murine IL-2 (Peprotech), 27.5 μM 2-Mercaptoehanol (Gibco), and 100 μg/mL of respective antibodies. After 48 hrs, CD8+ T cells were harvested and stained with anti-CD8a antibody (AF647, clone 53–6.7, BioLegend) and DAPI, and proliferation was analyzed by CFSE dilution using flow cytometry. After removal of CD8+ T cells, the viability of remaining PDAC cells were measured by CellTiter-Glo (Promega).
4
CFSE-Based Proliferation Assay for CD4+ T Cells
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For the proliferation assay, 1 × 106 purified CD4+CD25– T cells were labeled with the carboxyfluorescein succinimidyl ester (CFSE) (1 μM; Invitrogen, Carlsbad, CA, USA) in 1 mL pre-warmed phosphate-buffered saline, followed by incubation for 10 min at room temperature and neutralization with pre-chilled complete medium containing 10% fetal bovine serum. CFSE-labeled cells were re-suspended and cultured in complete medium in the presence of plate-bound anti-CD3 (coating concentration was 2.5 μg/mL, Biolegend, San Diego, CA, USA) and soluble anti-CD28 (2.5 μg/mL, Biolegend, San Diego, CA, USA) in 24-well flat-bottom plates for 72 h. After culture, the cells were collected for anti-TIGIT APC and anti-CD226 PerCP-CY5.5 staining and then analyzed to determine the CFSE intensities. Each experiment was performed and analyzed using a FACS JAZZ instrument.
5
MDSC Suppression of CD8+ T Cell Proliferation
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CD11b+Gr1+ MDSCs were sorted from tumors of tumor-bearing mice using an EasySep™ Mouse MDSC (CD11b+Gr1+) Isolation Kit. CD8+ T cells were isolated from the spleen of naive mice using an EasySep mouse CD8+T cell isolation kit (STEMCELL) according to the manufacturer’s instructions and were labeled with 1µM carboxyfluorescein succinimidyl ester (CFSE) (BioLegend). T cells were stimulated with anti-CD3 (1 µg/mL; Bio-Legend) and anti-CD28 (1 µg/mL; BioLegend), incubated at 105 cells per well, and co-cultured with sorted MDSCs at different ratios for 72 h. CD8+T cell proliferation by CFSE was measured using FACS analysis.
6
PDAC Organoids Potentiate CD8+ T Cell Killing
7
Comprehensive T Cell Proliferation Assay
8
T-cell Proliferation Assay
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CD4 or CD8 T cells were pre-loaded with CFSE as per manufacturer’s instruction (BioLegend) and then stimulated with CD3/CD28 antibodies or OVA peptides as described above for up to 72 h. T-cell proliferation as determined by quantification of the percentage of cells exhibiting CFSE dilution was detected by flow cytometry.
9
Quantifying T Cell Proliferation
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Isolated T cells were stained with carboxyfluorescein succinimidyl ester (CFSE; 2.5 million cells/ml, 5 μM CFSE, Biolegend) for 10 min at room temperature (r.t.) in the dark, before the reaction was stopped with cold medium containing 10% FCS. After washing the cells with medium, they were activated and stimulated as described above. Fluorescence intensity of the cells was quantified with a FACSCanto on day one (basis measurement) and day five.
10
Immune Response and Cell Cytotoxicity Assays
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Splenocytes isolated from WT and Ack1 KO mice were incubated with TRAMP-C2 cells. After 24 h, cells were labeled with 7-AAD (Biolegend) and the percentage of 7-AAD+ cells were evaluated by flow cytometry. In addition, splenocytes were isolated from C57BL/6 mice, treated overnight with 1 μM (R)-9b . Cells were washed once with PBS and incubated with TRAMP-C2 cells, pre-stained with CFSE (BioLegend). After 24 h, cells were labeled with 7-AAD (Biolegend) and cell lysis was evaluated using flow cytometry analyzing the percentage of CFSE+ 7-AAD+ cells.
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